Virus-like particles comprising a matrix protein from a plant enveloped virus and uses thereof

ABSTRACT

The present invention relates to novel virus-like particles (VLPs) comprising a matrix protein derived from a first plant enveloped virus and a surface polypeptide. The surface polypeptide comprises (a) a surface exposed portion derived from a target polypeptide (b) a transmembrane domain, and (c) a cytosolic tail derived from a transmembrane (e.g., glycoprotein) of a second plant enveloped virus. The target polypeptide may be antigenic or therapeutic. The first and the second plant enveloped viruses may be the same. Either plant enveloped virus may be a plant rhabdovirus. Also provided are methods of making and using the VLPs.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/614,141, filed Mar. 22, 2012, the content of which is incorporatedherein by its entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates generally to enveloped virus-likeparticles (VLPs) preferably produced in plants, and methods for makingand using the VLPs.

BACKGROUND OF THE INVENTION

Virus-like particles (VLPs) are complex structures formed byself-assembling viral proteins without the presence of the viral genome.The VLP platform is increasingly used to enhance target-specificimmunogenicity. VLPs can be produced in several expression systems,including plants. Production of VLPs in plants offers additionaladvantages, including safety and time efficiency. However, formation ofVLPs in plants is part of a physiological process and results information of particles of various sizes and shapes, making commercialmanufacturing and a smooth regulatory path highly challenging. The sizeof wild-type influenza virus particles depends on the strain and variesbetween 70 and 270 nm (Nayak et al., 2009, Virus Res 143(2):147-161).Plant-produced influenza viral particles (VLPs) have different shapesand a broad size range. For example, enveloped pleiomorphic influenzaVLPs have been reported to be 100 to 150 nm in size. (Vezina et al.,2011, BioPharm International Supplements 24(5):s27-s30). Thus, thereremains a need for more uniform VLPs suitable vaccine development.

SUMMARY OF THE INVENTION

The disclosed subject matter of the present invention relates to novelvirus-like particles (VLPs) having a matrix protein derived from anenveloped virus and a surface polypeptide and their uses. These VLPs areenveloped, and substantially uniform in size.

According to one aspect of the present invention, a virus-like particle(VLP) is provided. The VLP comprises a matrix protein derived from afirst plant enveloped virus and a surface polypeptide. The surfacepolypeptide comprises a surface exposed portion derived from a targetpolypeptide, a transmembrane domain, and a cytosolic tail. The cytosolictail is derived from a transmembrane protein (e.g., glycoprotein) of asecond plant enveloped virus. The VLP may be produced in a plant cell, aplant, or a portion of a plant.

The first and the second plant enveloped viruses may be the same ordifferent, preferably the same. Either may be a plant rhabdovirus ornon-rhabdovirus. Preferably, the first plant enveloped virus is a plantrhabdovirus. The plant rhabdovirus may be selected from the groupconsisting of Lettuce Necrotic Yellows virus (LNYV), Northern CerealMosaic virus (NCMV), Sonhus Virus (SonV) and Broccoli necrotic yellowsvirus (BNYV). Preferably, the plant rhabdovirus is Lettuce NecroticYellows virus (LNYV).

The surface exposed portion of the surface polypeptide may be anantigenic polypeptide, such as a vaccine component, or may be atherapeutic agent. The therapeutic agent may be a therapeuticpolypeptide.

The target polypeptide may be derived from a pathogen. The pathogen maybe selected from the group consisting of a virus, a bacterium, aparasite and a fungus. The virus may be an animal virus. The animalvirus may be selected from the group consisting of an influenza virus, arespiratory syncytial virus (RSV), a human immunodeficiency virus (HIV),a hepatitis B virus (HBV), a hepatitis C virus (HCV), a humanpapillomavirus (HPV), an Ebola virus, a Yellow fever virus, a rotovirus,and a vesicular stomatitis virus (VSV).

The target polypeptide may be a native surface polypeptide. The nativesurface polypeptide may be a hemagglutinin of an influenza virus.

The target polypeptide may be an artificial surface polypeptide. Theartificial surface polypeptide may be protective antigen 83 (PA83) fromBacillus anthracis, Pfs25 from Plasmodium falciparum or other solubleprotein or peptide.

The influenza virus may be selected from the group consisting of anInfluenza A virus and an Influenza B virus. The influenza virus may beselected from the group consisting of Influenza A Indonesia 05/05strain, Influenza A virus California/04/2009 (H1N1) strain, InfluenzaA/Victoria/3/75 (H3N2) strain and Influenza B Hong Kong/330/2001.

The target polypeptide may be a therapeutic agent. The therapeutic agentmay be a therapeutic polypeptide.

The transmembrane domain may be native or foreign to the surface exposedportion of the surface polypeptide. The transmembrane domain may bederived from a cellular or viral transmembrane protein. The viraltransmembrane domain may be derived from the transmembrane protein fromwhich the cytosolic tail is derived.

According to another aspect of the present invention, a method ofproducing virus-like particles (VLPs) in a plant cell, a plant, or aportion of a plant is provided. The method comprises introducing one ormore nucleic acid molecules into the plant cell, the plant, or theportion of a plant. The one or more nucleic acid molecules comprise afirst nucleotide sequence encoding a matrix protein derived from a firstplant enveloped virus and a second nucleotide sequence encoding asurface polypeptide. The surface polypeptide comprises a surface exposedportion derived from a target polypeptide, a transmembrane domain, and acytosolic tail. The cytosolic tail is derived from a transmembraneprotein (e.g., glycoprotein) of a second plant enveloped virus. Thefirst and second plant enveloped viruses may be the same or different,preferably the same. Either may be a plant rhabdovirus ornon-rhabdovirus. Preferably, the first plant enveloped virus is a plantrhabdovirus. The method further comprises maintaining the plant cell,the plant, or the portion of a plant under conditions permittingco-expression of the matrix protein and the surface polypeptide suchthat the VLPs are produced. The VLPs may be substantially uniform insize. The method may further comprise purifying the VLPs from the plantcell, the plant or the portion of the plant.

The one or more nucleic acid molecules may be introduced into the plantcell, the plant or the portion of a plant by infiltration, particlebombardment, or inoculation. The one or more nucleic acid molecules maybe introduced into the plant or a portion thereof transiently or stably.

Various immunogenic compositions are provided. In some embodiments, theimmunogenic composition comprises an effective amount of the VLPs of thepresent invention, and the VLPs are substantially uniform in size. Inother embodiments, the immunogenic composition comprises an effectiveamount of the VLPs produced by the method of the present invention, andthe VLPs are substantially uniform in size. The immunogenic compositionmay further comprise an adjuvant and/or an excipient.

A method of inducing an immune response to the target polypeptide in asubject is provided. The method comprises administering to the subjectan effective amount of the immunogenic composition of the presentinvention, wherein the VLPs are substantially uniform in size.

A method of inducing a protective immune response to a pathogen in asubject is also provided. The method comprises administering to thesubject an effective amount of the immunogenic composition of thepresent invention, wherein the VLPs are substantially uniform in size.The target polypeptide is derived from the pathogen. The pathogen may bean influenza virus. The target polypeptide may be derived from ahemagglutinin.

A recombinant plant cell comprising one or more nucleic acid moleculesis further provided. The one or more nucleic acid molecules comprise afirst nucleotide sequence encoding a matrix protein derived from a firstplant enveloped virus and a second nucleotide sequence encoding asurface polypeptide. The surface polypeptide comprises (a) a surfaceexposed portion derived from a target polypeptide, (b) a transmembranedomain, and (c) a cytosolic tail derived from a transmembrane protein(e.g., glycoprotein) of a second plant enveloped virus. The first andsecond plant enveloped viruses may be the same or different, preferablythe same. Either may be a plant rhabdovirus or non-rhabdovirus.Preferably, the first plant enveloped virus is a plant rhabdovirus. Alsoprovided is a plant or a portion thereof comprising the plant cell ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows (A) an electron microscopy image and (B) the organizationof a plant rhabdovirus.

FIG. 2 is a diagram illustrating the genome organization of the T-DNAregion of a miniBYV vector for co-expression of two target proteins,i.e., Target 1 and Target 2, each operatively linked to a regulatoryregion. L-Pro, papain like leader proteinase; Met, Hel and Pol,methyltransferase, RNA helicase, RNA-dependent RNA polymerase domains ofthe replicase, respectively; 2Enx35S, 35S promoter with dual enhancersfrom Cauliflower mosaic virus; NOS—Nopaline synthase terminator; LB andRB, left and right borders of the T-DNA, respectively.

FIG. 3 is a diagram illustrating the genome organization of the T-DNAregion of a miniBYV-HAi-TM/M_(LNYV) or miniBYV-HAi-TM/M_(NCMV) vectorfor co-expression of an HAi-TM protein and a matrix protein of LettuceNecrotic Yellows virus (LNYV) (M_(LNYV)) or Northern Cereal Mosaic virus(NCMV) (M_(NCMV)), respectively. L-Pro, papain like leader proteinase;Met, Hel and Pol, methyltransferase, RNA helicase, RNA-dependent RNApolymerase domains of the replicase, respectively; 2Enx35S; 35S promoterwith dual enhancers from Cauliflower mosaic virus; NOS—Nopaline synthaseterminator; LB and RB, left and right borders of the T-DNA,respectively.

FIG. 4 is a diagram illustrating the genome organization of the T-DNAregion of a miniBYV-HAi-TM(G)/M_(LNYV) vector for co-expression of anHAi-TM(G)_(LNYN) protein and an M_(LNYV) protein. L-Pro, papain likeleader proteinase; Met, Hel and Pol, methyltransferase, RNA helicase,RNA-dependent RNA polymerase domains of the replicase, respectively;2Enx35S, 35S promoter with dual enhancers from Cauliflower mosaic virus;NOS—Nopaline synthase terminator; LB and RB, left and right borders ofthe T-DNA, respectively.

FIG. 5 depicts a representative western blot for time course analysis ofHAi-TM expression detected by an anti-HAi 5G6 primary monoclonalantibody after infiltration. Reference HAi in the amount of 60, 30, or15 ng was used as standard for quantification. Lanes 1 and 2, manualinfiltrated N. benthamiana; 3 and 4, vacuum infiltrated N. benthamiana.

FIG. 6 shows (A) distribution of HAi-TM VLPs through a 10%-40% sucrosegradient and (B) morphology of the HAi-TM VLPs from fraction 10 of (A)detected by transmission electron microscopy (TEM) negative staining.

FIG. 7 shows (A) distribution of HAi-TM/M1 VLPs through a 10-40% sucrosegradient; and (B) morphology of the HAi-TM/M1 VLPs from fraction 10 of(A) detected by TEM negative staining.

FIG. 8 shows (A) expression of the influenza A/Indonesia/05/2005 matrix(M1) protein in leaf samples (lanes 1-3, different replicas from theinfiltrated plants producing HAi-TM/M1 VLPs); and (B) absence of the M1protein from the HAi-TM/M1 VLPs in the 10-40% sucrose gradient fractionsas shown on FIG. 7A. Std, M1 protein standard.

FIG. 9 shows (A) distribution HAi-TM/M_(LNYV) VLPs in a 10-40% sucrosegradient and (B) immunogold labeling of the HAi-TM/M_(LNYV) VLPs with ananti-HAi 5G6 mouse monoclonal antibody.

FIG. 10 shows (A) distribution of HAi-TM(G)/M_(LNYV) VLPs through a10%-40% sucrose gradient and (B) immunogold labeling of theHAi-TM(G)-M_(LNYV) VLPs from fraction 7 of (A) with an anti-HAi 5G6mouse monoclonal antibody.

FIG. 11 shows the amino acid sequences of (A) a hemagglutinin of H5N1avian influenza virus (A/Indonesia/05/2005) (HAi-TM) (SEQ ID NO: 1)having a PR1a Nicotiana tabacum signal peptide (underlined), an HAiectodomain, an HAi transmembrane domain (bold), and an HAi cytosolictail (italicized), and (B) a hemagglutinin of H5N1 avian influenza virus(A/Indonesia/05/2005) (HAi-TM(G)_(LNYV)) (SEQ ID NO: 2) having a PR1a N.tabacum signal peptide (underlined), an HAi ectodomain, a transmembranedomain of an LNYV glycoprotein (bold), and a cytosolic tail of an LNYVglycoprotein (italicized).

FIG. 12 shows the amino acid sequences of (A) a matrix protein of LNYV(M_(LNYV)) (SEQ ID NO: 3) and (B) a matrix protein of H5N1 avianinfluenza virus (A/Indonesia/05/2005) (M1 protein) (SEQ ID NO: 4).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that novel virus-likeparticles (VLPs) comprising a matrix protein derived from a plantenveloped virus and a surface polypeptide can be produced in plants.These VLPs are enveloped, and are substantially uniform in size, and maybe used to design and manufacture effective human vaccines.

The term “protein” used herein refers to a biological moleculecomprising amino acid residues. A protein may comprise one or morepolypeptides. Each polypeptide may be a subunit of a protein. Theprotein may be in a native or modified form, and may exhibit abiological function when its polypeptide or polypeptides are properlyfolded or assembled.

The term “polypeptide” used herein refers to a polymer of amino acidresidues with no limitation with respect to the minimum length of thepolymer. Preferably, the polypeptide has at least 4 amino acids. Apolypeptide may be a full-length protein, or a fragment or variantthereof.

The term “fragment” of a protein as used herein refers to a polypeptidehaving an amino acid sequence that is the same as a part, but not all,of the amino acid sequence of the protein. Preferably, a fragment is afunctional fragment of a protein that retains the same function as theprotein.

The term “variant” of a protein used herein refers to a polypeptidehaving an amino acid sequence that is the same as that of the proteinexcept having at least one amino acid modified, for example, deleted,inserted, or replaced, respectively. The amino acid replacement may be aconservative amino acid substitution, preferably at a non-essentialamino acid residue in the protein. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains are known in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), non-polar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). A variant of a protein may havean amino acid sequence at least about 80%, 90%, 95%, or 99%, preferablyat least about 90%, more preferably at least about 95%, identical to theamino acid sequence of the protein. Preferably, a variant is afunctional variant of a protein that retains the same function as theprotein.

The term “derived from” used herein refers to an origin or source, andmay include naturally occurring, recombinant, unpurified or purifiedmolecules. The molecules of the present invention may be derived fromviral or non-viral molecules. A protein or polypeptide derived from anoriginal protein or polypeptide may comprise the original protein orpolypeptide, in part or in whole, and may be a fragment or variant ofthe original protein or polypeptide.

The term “native” used herein refers to a molecule (e.g., a protein orpolypeptide) that is naturally occurring. The term “artificial” usedherein refers to a molecule (e.g., a protein or polypeptide) that is notnaturally occurring, but synthesized artificially, for example,recombinantly or chemically.

According to one aspect of the present invention, a virus-like particle(VLP) is provided. The VLP comprises a matrix protein and a surfacepolypeptide. The matrix protein is derived from a first plant envelopedvirus, preferably plant rhabdovirus, more preferably, lettuce necroticyellows virus. The surface polypeptide comprises a surface exposedportion derived from a target polypeptide, a transmembrane domain and acytosolic tail. The cytosolic tail is derived from a transmembraneprotein (e.g., glycoprotein) of a second plant enveloped virus. Forexample, the cytosolic tail may be derived from a rhabdoviralglycoprotein. The transmembrane domain and the cytosolic tail may bederived from the same or different plant enveloped viruses, preferablyfrom the same plant enveloped virus. In one embodiment, thetransmembrane domain is derived from a target protein while thecytosolic tail is derived from a rhabdovirus glycoprotein. In anotherembodiment, the transmembrane domain and the cytosolic tail are bothderived from the same plant rhabdoviral glycoprotein. The matrixprotein, the transmembrane domain and the cytosolic tail may be derivedfrom the same plant enveloped virus.

The VLP may be produced in a plant cell or other cells. The plant cellmay be a cell in a plant, a plant part (e.g., leaf, stem, root, floraltissue, seed or petiole), or a cell culture medium. The plant may be awhole growing plant. Preferably, the plant cell is in a plant leaf. Thecell culture media may be any media suitable for growing plant cells,preferably in suspension. The plant cell is preferably suitable forexpression of a VLP. For example, the plant cell may be in N.benthamiana leaves. Other suitable plants include Nicotiana clevelandii,Beta vulgaris, Spinacia oleracea, Brassica spp, Lactuca sativa, Pisumsativum, Nicotiana tabacum, Plantago lanceolata, Tetragoniatetragonioides, Montia perfoliata, Stellaria media, Medicago truncatula,and Chenopodium foliosum.

The VLP may comprise a membrane. The surface polypeptide of the VLP maybe integrated into the membrane. The membrane may be derived from thecell in which the VLP is assembled, made or produced.

The matrix protein may be derived from a natural matrix protein of anyplant enveloped virus. The matrix protein may have an amino acidsequence at least about 80%, 85%, 90%, 95%, or 99%, preferably at leastabout 90%, more preferably at least about 95%, further preferably atleast about 99%, most preferably about 100%, identical to that of thecorresponding natural matrix protein.

The surface exposed portion derived from a target polypeptide is aportion of the surface polypeptide that is on the surface of the VLP.The surface polypeptide may be an antigenic polypeptide, such as avaccine component, or may be a therapeutic agent. The therapeutic agentmay be a therapeutic polypeptide. The surface polypeptide, either in theVLP or purified from the VLP, may be used for various purposes. Forexample, the surface polypeptide may be antigenic and used to induce animmune response in a subject when introduced into the subject. It mayalso be used as a therapeutic agent to treat a disease or disorder in asubject when administered to the subject. It may further be used as adiagnostic agent for diagnosis of a disease or disorder in a subjectwhen used in testing a sample from the subject suspected of having thedisease or disorder.

The surface polypeptide may be derived from an antigenic targetpolypeptide, and capable of inducing an immune response in a subjectwhen introduced into the subject, and may be used to form or become avaccine candidate. It may comprise one or more epitopes (linear and/orconformational) capable of stimulating the immune system of a subject tomake a humoral and/or cellular antigen-specific immune response. Ahumoral immune response refers to an immune response mediated byantibodies produced by B lymphocytes, or B cells, while a cellularimmune response refers to an immune response mediated by T lymphocytes,or T cells, and/or other white blood cells. In general, a B-cell epitopecontains at least about 5 amino acids but can be 3-4 amino acids, whilea T-cell epitope includes at least about 7-9 amino acids and a helperT-cell epitope includes at least 12-20 amino acids. The targetpolypeptide may be derived from, in part or in whole, a natural protein(e.g., a surface protein or toxin subunit) of a pathogenic organism orpathogen. The target polypeptide may comprise an amino acid sequence atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or99%, preferably at least about 90%, more preferably at least about 95%,further preferably at least about 99%, most preferably about 100%,identical to that of the corresponding natural pathogenic protein. Thepathogen may be an intracellular or extracellular pathogen. Exemplarypathogens include viruses, bacteria, parasites and fungi. The virusesmay be animal or plant viruses, preferably animal viruses. An animalvirus may be selected from the group consisting of an influenza virus, arespiratory syncytial virus (RSV), a human immunodeficiency virus (HIV),a hepatitis B virus (HBV), a hepatitis C virus (HCV), a humanpapillomavirus (HPV), an Ebola virus, a Yellow fever virus, a rotovarus,and a vesicular stomatitis virus (VSV). Preferably, the targetpolypeptide is derived from an influenza virus.

The target polypeptide may be a native surface polypeptide or anartificial surface polypeptide. The native surface polypeptide may be ahemagglutinin of an influenza virus. The artificial surface polypeptidemay be protective antigen 83 (PA83), Pfs25, or other soluble protein orpeptide. The PA83 is from Bacilus anthracis. The Pfs25 is fromPlasmodium falciparum.

An influenza virus may be selected from the group consisting of anInfluenza A virus and an Influenza B virus. The influenza virus may beselected from the group consisting of Influenza A Indonesia 05/05strain, Influenza A virus California/04/2009 (H1N1) strain, InfluenzaA/Victoria/3/75 (H3N2) strain and Influenza B Hong Kong/330/2001.

The transmembrane domain of the surface polypeptide may be a nativeprotein or an artificial protein. The transmembrane domain may be nativeor foreign to the surface exposed portion of the surface polypeptide orthe target polypeptide. The transmembrane domain may be derived from atransmembrane protein, preferably the same transmembrane protein, fromwhich the matrix protein and the cytosolic tail are derived. Thetransmembrane domain may be derived from a cellular or viraltransmembrane protein, or a surface protein. The transmembrane domainmay or may not be derived from the target polypeptide. The viraltransmembrane protein may be a transmembrane protein from a plant virus.The plant virus may be a plant enveloped virus, preferably the plantenveloped virus from which the matrix protein and the cytosolic tail arederived. The transmembrane domain may be derived from a glycoprotein ofa virus, preferably a glycoprotein of a plant enveloped virus, morepreferably a glycoprotein of the plant enveloped virus from which thematrix protein and the cytosolic tail are derived.

A plant enveloped virus has a lipid envelope. This lipid envelope istypically derived from the membrane of a host cell from which the virusparticle buds off. The plant enveloped virus may be a plant rhabdovirusor non-rhabdovirus. Examples of plant rhabdoviruses include LettuceNecrotic Yellows virus (LNYV), Northern Cereal Mosaic virus (NCMV),Sonhus Virus (SonV) Broccoli necrotic yellows virus (BNYV), and otherplant rhabdoviruses recognized by the International Committee onTaxonomy of Viruses (ICTV). Preferably, the plant enveloped virus isLettuce Necrotic Yellows virus (LNYV).

LNYV and NCMV rhabdoviruses are characterized by monocistronic negativesense RNA and belong to the family Rhabdoviridae and the genusCytorhabdoviruses. Plant rhabdoviruses are characterized by thebacilli-form structure (FIG. 1A) and are about 130-350 nm in length andabout 40-100 nm in width (Jackson et al, 2005, Ann. Rev. Phytopathol.43:623-660). The two proteins of the virus that are key for assembly ofbacilli-form particles are matrix protein (M protein) and glycoprotein(G protein). M protein forms a layer inside the virion's envelope andinteracts with the cytosolic tale of G protein (FIG. 1B).

According to another aspect of the present invention, a method forproducing the VLPs of the present invention in a plant cell, a plant, ora portion of a plant is provided. The method comprises introducing oneor more nucleic acid molecules into the plant cell, the plant, or theportion of a plant. The one or more nucleic acid molecules comprise afirst nucleotide sequence encoding the matrix protein and a secondnucleotide sequence encoding the surface polypeptide. The matrix proteinis derived from a first plant enveloped virus. The surface polypeptidecomprises a surface exposed portion derived from a target polypeptide, atransmembrane domain, and a cytosolic tail. The cytosolic tail isderived from a transmembrane protein (e.g., glycoprotein) of a secondplant enveloped virus. For example, the cytosolic tail may be derivedfrom a plant rhabdoviral glycoprotein. The transmembrane domain may ormay not be derived from the target polypeptide. The first and the secondplant enveloped viruses may be the same or different, preferably thesame. Either may be a plant rhabdovirus or non-rhabdovirus. Preferably,the first enveloped plant virus is a plant rhabdovirus. The methodfurther comprises maintaining the plant cell, the plant, or the portionof a plant under conditions permitting co-expression of the matrixprotein and the surface polypeptide such that the VLPs are produced.

The VLPs produced by a method according to the present invention may beof any sizes. For example, the VLPs may have a diameter in the range ofabout 5-350 nm, including about 40-60 nm. Preferably, the VLPs aresubstantially uniform in size. The term “substantially uniform in size”used herein means that at least about 50%, 60%, 70%, 80%, 90%, 95% or99%, preferably at least about 80%, more preferably at least about 90%,most preferably at least about 95%, of the VLPs have a diameter withinless than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%,preferably less than about 30%, more preferably less than about 20%,most preferably less than 10%, of the average diameter of all the VLPs.The diameter of a VLP may be the average or mean value of one or moremeasurements of the diameter of the VLP. The diameter of a VLP may bedetermined by conventional techniques known in the art, for example, byobservation using an electron microscopy, sucrose gradientfractionation, size exclusion chromatography, and dynamic lightscattering. The diameter values of the VLPs may be analyzed usingvarious software, for example, ImageJ software (NIH, Bethesda, Md.) andother commercially available software.

The one or more nucleic acid molecules may further comprise a regulatoryregion operatively linked to the first or second nucleotide sequence.The regulatory region may be activated in a plant cell for theexpression of the protein encoded by the first or the second nucleotidesequence. The regulatory region may include a promoter, for example, aplant viral promoter. Preferably, the first and second nucleotidesequences are in the same nucleic acid molecule, but linked to separateregulatory regions. The one or more nucleic acid molecules may furthercomprise a third nucleotide sequence encoding an additional surfacepolypeptide.

For each nucleic acid molecule, a vector comprising the nucleotidesequence of the nucleic acid molecule is provided. The vector mayinclude a border sequence of a bacterial transfer DNA at either end,situated in a bacterial transfer DNA, to allow for delivery of thenucleic acid into a plant cell. Specifically, the vector may compriseone or more nucleic acid sequences derived from a Ti plasmid of a binaryvector, and may also comprise a plant viral sequence. Such a vector,including elements of a Ti plasmid and a viral vector, is also called alaunch vector. This vector may also be used for co-expression of theprotein or polypeptide of interest (e.g., the matrix protein and/or thesurface polypeptide) with a protein such as a silencing suppressor or apost-translational modifying enzyme such as PNGaseF, to modify, affectexpression and/or increase production of the protein of interest by, forexample, facilitating maturation or accumulation of the protein.

Each nucleic acid molecule may be introduced into the plant cell, theplant, or the portion of a plant (e.g., leaf, stem, root, floral tissue,seed or petiole) using techniques known in the art. For example, thenucleic acid molecule may be delivered via infiltration, particlebombardment, or inoculation. The nucleic acid molecule could be used asa part of an inducible system activated by, for example, chemical, lightor heat shock. Preferably, the nucleic acid molecule is introduced intothe plant cell via infiltration. The nucleic acid molecule may beintroduced transiently or stably.

A recombinant plant cell comprising one or more nucleic acid moleculesis provided. The one or more nucleic acid molecules comprise a firstnucleotide sequence encoding a matrix protein and a second nucleotidesequence encoding a surface polypeptide. Preferably, the first andsecond nucleotide sequences are in one nucleic acid molecule. The matrixprotein is derived from a first plant enveloped virus. The surfacepolypeptide comprises a surface exposed portion derived from a targetpolypeptide, a transmembrane domain and a cytosolic tail. The cytosolictail is derived from a transmembrane protein (e.g., glycoprotein) of asecond plant enveloped virus. The first and second plant envelopedviruses may be the same or different, preferably the same. Either may bea plant rhabdovirus or non-rhabdovirus. Preferably, the first plantenveloped virus is a plant rhabdovirus. The transmembrane domain may ormay not be derived from the target polypeptide. The cytosolic tail maybe derived from a glycoprotein of a plant enveloped virus. For example,the cytosolic tail may be derived from a rhabdoviral glycoprotein. Thefirst or second nucleotide sequence may be operatively linked to apromoter capable of being activated in the plant cell for co-expressionof the matrix protein or the surface polypeptide, respectively. Alsoprovided is a plant or a portion thereof comprising the plant cell ofthe present invention.

For production of the VLPs of the present invention, a plant cell, or aplant or a portion thereof may be maintained under conditions permittingco-expression of the matrix protein and the surface polypeptide,resulting in the production of the VLPs. Such conditions includesuitable temperature, humidity, pressure, light/dark cycle, andillumination. Preferably, the matrix protein and the surface polypeptideare co-expressed in the same plant cells.

The production method may further comprise purifying the VLPs.Conventional purification techniques known in the art may be used. Forexample, the VLP may be purified from the plant cell using an antibodyor a receptor capable of binding the surface polypeptide. Thepurification process may comprise extraction of the VLPs from the plantcell using an extraction buffer. After low speed centrifugation,supernatant may be clarified by filtration and used for chromatography.The purified VLPs may be at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95% or 99%, preferably at least about 50%, morepreferably at least about 75%, most preferably at least about 95%, pure.The VLPs in a crude plant extract may be used.

An immunogenic composition is provided. The composition comprises aneffective amount of the VLPs of the present invention. Preferably, theVLPs are substantially uniform in size. The effective amount of the VLPsis an amount of the VLPs sufficient to make the composition immunogenic,i.e., capable of inducing an immune response in a subject whenintroduced into the subject. The composition may further comprise anadjuvant and/or an excipient. The immunogenic compositions may be usedto induce an immune response in a subject. Several methods of inducingan immune response are provided.

A method of inducing an immune response to the target polypeptide in asubject is provided. The method comprises administering to the subjectan effective amount of the immunogenic composition of the presentinvention. Preferably, the VLPs are substantially uniform in size. Theimmunogenic composition may be administered in an appropriate number ofdoses.

A method of inducing a protective immune response to a pathogen in asubject is also provided. The method comprises administering to thesubject an effective amount of the immunogenic composition of thepresent invention. Preferably, the VLPs are substantially uniform insize. The target polypeptide is derived from the pathogen. For example,the target polypeptide may be a hemagglutinin and the pathogen is aninfluenza virus.

The subject may be an animal, including a mammal, for example, a human,a mouse, a cow, a horse, a chicken, a dog, a cat, and a rabbit. Theanimal may be an agricultural animal (e.g., horse, cow and chicken) or apet (e.g., dog and cat). The subject is preferably a human or a mouse,more preferably a human. The subject may be a male or female. Thesubject may also be a newborn, a child or an adult. The subject may havesuffered or be predisposed to a disease or medical condition, which maybe caused by or associated with a pathogen.

The immunogenic composition may be formulated, for example, for oral,sublingual, intranasal, intraocular, rectal, transdermal, mucosal,topical or parenteral administration. Parenteral administration mayinclude intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intranasal,intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p.),intra-arterial, intramedulary, intracardiac, intra-articular (joint),intrasynovial (joint fluid area), intracranial, intraspinal, andintrathecal (spinal fluids) administration. Any device suitable forparenteral injection or infusion of the composition may be used for suchadministration.

The term “an effective amount” refers to an amount sufficient to achievea stated goal, and may vary depending on the stated goal and otherfactors. For example, an effective amount of VLPs in an immunogeniccomposition or an effective amount of an immunogenic compositioncomprising VLPs may vary depending on the physical characteristics ofthe subject, the nature and severity of the need of the VLPs, theexistence of related or unrelated medical conditions, the nature of theVLPs, the means of administering the VLPs to the subject, and theadministration route. A specific dose for a given subject may generallybe set by the judgment of a physician. The immunogenic composition maybe administered to the subject in one or multiple doses.

Various medicaments comprising the VLPs of the present invention areprovided. The VLPs are preferably substantially uniform in size. Themedicaments may comprise suitable adjuvants. In some embodiments, themedicaments are useful for inducing an immune response to the targetpolypeptide in a subject, and comprise an effective amount of the VLPsof the present invention. In some other embodiments, the medicaments areuseful for inducing a protective immune response to a pathogen in asubject, and comprise an effective amount of the VLPs of the presentinvention, wherein the target polypeptide is derived from the pathogen.For example, the target polypeptide may be a hemagglutinin and thepathogen is an influenza virus.

For each medicament of the present invention, a method for preparing themedicament is provided. The preparation method comprises admixing theVLPs of the present invention with a pharmaceutically acceptable carrierand/or excipient. The VLPs are preferably substantially uniform in size.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a percentage, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate.

Example 1 Construction of Mini-BYV Vectors

To demonstrate the feasibility of assembling VLPs using matrix proteins(M proteins) from different plant enveloped viruses, a rhabdoviral Mprotein and a target antigen containing a transmembrane (TM) domain anda cytosolic tail of a LNYV glycoprotein (G protein) were engineered forco-expression in plant cells. For co-expression of these proteins, aminiBYV vector was used (FIG. 2). The miniBYV vector contains aminireplicon derived from a Closteroveridae virus (e.g., Beet yellowsvirus), which comprises a nucleic acid sequence encoding only proteinsrequired for replication of the Closteroveridae virus. Specifically, theM protein from LNYV or NCMV was co-expressed with a hemagglutinin (HAi)from H5N1 avian influenza virus (A/Indonesia/05/2005).

In brief, a gene encoding a HAi protein having its native TM domain(HAi-TM) was cloned into a miniBYV vector to generate the miniBYV-HAi-TMplasmid. The HAi-TM protein (SEQ ID NO: 1) included a PR1a Nicotianatabacum signal peptide (underlined), an HAi ectodomain, an HAi nativetransmembrane domain (bold), and an HAi native cytosolic tail(italicized) (FIG. 11A). Subsequently, a gene encoding the M protein ofLNYV (M_(LNYV) protein) or NCMV (M_(NCMV) protein) was introduced intothe miniBYV-HAi-TM plasmid under the control of the closteroviralheterologous coat protein (CP) promoter to generate aminiBYV-Hai-TM/M_(LNYV) or miniBYV-Hai-TM/M_(NCMV) plasmid (FIG. 3). ForVLP evaluation, positive clones were transformed into Agrobacteriumtumefaciens strain GV3101 and then introduced into Nicotiana benthamianaplants to produce VLPs.

In parallel, the HAi gene was modified to encode a recombinant ahemagglutinin protein (HAi-TM(G)), in which the native TM and thecytosolic tail of HAi were replaced with the transmembrane domain andcytosolic tail from a glycoprotein of LNYV, respectively. A nucleotidesequence encoding the HAi-TM(G) protein (SEQ ID NO: 2), including a PR1aNicotiana tabacum signal peptide (underlined), an HAi ectodomain, anLNYV glycoprotein transmembrane domain (bold), and an LNYV glycoproteincytosolic tail (italicized) (FIG. 11B), was introduced into the miniBYVvector containing a nucleotide sequence encoding the M protein of LNYV(M_(LNYV) protein) (SEQ ID NO: 3) (FIG. 12A). The resulting construct,miniBYV-HAi-TM(G)/M_(LNYV) (FIG. 4), was sequenced, and positive cloneswere transformed into A. tumefaciens and then introduced into Nicotianabenthamiana plants to produce HAi-TM(G)-M_(LNYV) VLPs.

As a control, a gene encoding the matrix protein (M1 protein) of H5N1avian influenza virus (A/Indonesia/05/2005) (SEQ ID NO: 4) (FIG. 12B)was introduced into the minBYV-HAi-TM plasmid under the control of theclosteroviral heterologous coat protein (CP) promoter to generate theplasmid miniBYV-HAi-TM/M1. As described above, PR1a was used as a signalpeptide. Positive clones were transformed into A. tumefaciens and thenintroduced into Nicotiana benthamiana plants to produce HAi-TM/M1 VLPs.

Example 2 Optimization of Infiltration Procedure to Express Hai

To evaluate the expression level of HAi and develop vacuum infiltrationconditions, hydroponically grown N. benthamiana plants were used. Toanalyze the expression of HAi-TM, manual infiltration and vacuuminfiltration of miniBYV-HAi-TM were performed using the same buffer.Five week old Nicotiana benthamiana plants grown in Rockwool in clamshells were used for vacuum and manual infiltration. Agrobacteria weregrown in LB media, which was supplemented with 50 ng/ml Kanamycin and 50ng/ml Hygromycin. One liter overnight cultures were grown at 28° C.,shaking at 220 rpm for 18-24 hours.

Overnight concentration was determined by measuring the OD₆₀₀ for eachculture. The agrobacteria were pelleted by spinning at 4000 g for 15 minat 4° C. Bacterial pellets were re-suspended in 100 ml of fresh MMAmedia (10 mM MgCl2; 10 mM MES, pH 5.85; and 20 μM acetosyringone). Thefinal concentration for each culture was recorded after rocking for 2hours at room temperature. Cultures containing miniBYV and P1/HcProsilencing suppressor (miniBYV requires use of a silencing suppressor toachieve good target expression levels) were mixed to ratios of OD₆₀₀,1.0:0.2, respectively. For vacuum infiltration, clamshells wereinfiltrated with miniBYV:P1/HcPro containing agrobacteria. Infiltratedplants were fed 50 ppm hydrosol and placed in a growth room for 5-8days.

For manual infiltration, 3-5 leaves of hydroponically grown Nicotianabenthamiana were manually infiltrated using a 10 cc syringe without aneedle. Plants were fed with 50 ppm hydrosol and kept in the postinfiltration room for 5-8 days.

HAi-TM expression levels were determined based on the amount of HAi-TMprotein detected by an anti-HAi 5G6 monoclonal antibody in Western blotanalysis (FIG. 5). HAi-TM expression was higher in vacuum infiltratedleaves at 6 days post infiltrations (dpi) and 7 dpi compared to manuallyinfiltrated leaves (Table 1). For example, at 6 dpi, the HAi-TM proteinwas expressed at 249 mg/kg in vacuum infiltrated leaves, representing a43% increase in expression compared to manually infiltrated leaves.

TABLE 1 Time course expresssion levels for HAi-TM Days Manual PostInfiltration Infiltration (mg/kg) Vacuum Infiltration (mg/kg) 5 73 46 6174 249 7 120 123 8 180 105

Example 3 Plant-Produced HAi-TM VLPs

To characterize VLPs formation HAi-TM were cloned in to miniBYV vectorusing PacI/NheI restriction sites. The resulting plasmid miniBYV HAi-TMwas transformed in to GV3101 Agrobacterium strain. Nicothianabenthamiana infiltrated leaves were harvested at 7 days postinfiltration and VLPs were purified. Distribution of HAi-TM VLPs withoutany matrix protein through a 10%-40% sucrose gradient with peakfractions 9 and 10 was detected by an anti-HAi 5G6 mouse monoclonalantibody (developed by the Immunology group at Fraunhofer—USA Center forMolecular Biotechnology) (FIG. 6A). To evaluate the morphology ofHAi-VLPs in fraction 10, VLPs from this fraction were analyzed bynegative staining using transmission electron microscopy (TEM). HAi-TMVLPs with different sizes and shapes were observed (FIG. 6B).

HAi-TM/M1 VLPs having HAi protein with its native transmembrane domainand cytosolic tail coexpressed with M1 of influenza A/Indonesia/05/05were produced by expressing miniBYV-HAi-TM/M1 in plants using vacuuminfiltration as described in Example 2. HAi-TM/M1 VLPs were purifiedusing a sucrose gradient, and characterized by Western blot analysis andelectron microscopy using the anti-HAi 5G6 mouse monoclonal antibody,and were found to distribute through a 10%-40% sucrose gradient withpeak fractions 9 and 10 (FIG. 7A). No M1 protein incorporation wasdetected in the same HAi-TM/M1 VLPs through the 10-40% sucrose gradient(FIG. 8A) using a polyclonal goat anti-M1 antibody from Meredian Lifescience, Inc, USA. Meanwhile, the expression of M1 protein was confirmedby western blot using a goat anti-M1 protein polyclonal antibody (FIG.8B). No uniformed VLPs were detected after co-expression HAi-TM andInfluenza matrix M1 protein (FIG. 7B).

Example 4 Plant-Produced HAi-TM/M_(LNYV) VLPs

HAi-TM/M_(LNYV) VLPs having HAi-TM protein (i.e., HAi protein with itsnative transmembrane domain and cytosolic tail) and LNYV M protein(M_(LNYV) protein) were produced by expressing miniBYV-HAi-TM/M_(LNYV)in plants using vacuum infiltration as described in Example 2.HAi-TM/M_(LNYV) VLPs were purified using a 10%-40% sucrose gradient andcharacterized by SDS-PAGE, Western blot analysis and electronmicroscopy. The presence of the HAi immunological determinant on thesurface of these VLPs was confirmed by immunogold labeling using theanti-HAi 5G6 mouse monoclonal antibody (FIG. 9B). Also, this antibodywas used to detect VLPs distribution in a 10-40% sucrose gradient with apeak in fraction 10 (FIG. 9A). The distribution of HAi-TM/M_(LNYV) VLPswas similar to that observed for HAi-TM/M1 VLPs (FIGS. 7A and 9A). Therewas no noticeable difference in the VLP morphology (e.g., shape andsize) (FIGS. 7B and 9B).

Example 5 Plant-Produced Uniform HAi-TM(G)/M_(LNYV) VLPs

HAi-TM(G)/M_(LNYV) VLPs having HAi-TM(G) protein (i.e., HAi protein witha transmembrane domain and a cytosolic tail from a rhabdoviralglycoprotein (G) protein) and LNYV M protein (M_(LNYV) protein) wereproduced by expressing miniBYV-HAi-TM(G)/M_(LNYV) in plants using vacuuminfiltration as described in Example 2. After separation ofHAi-TM(G)/M_(LNYV) VLPs by a 10%-40% sucrose gradient, 1 mL fractionswere collected and analyzed by Western blot analysis using the anti-HAi5G6 monoclonal antibody. A shift in the target collection peak wasobserved. Unlike HAi-TM/M_(LNYV) VLPs, the HAi-TM(G)/M_(LNYV) VLPs weredetected in fractions 6, 7 and 8 with a sharp peak in fraction 7 (FIG.10A). Immunogold labeling following TEM showed round-shapedHAi-TM(G)/M_(LNYV) VLPs (FIG. 106). The size of the HAi-TM(G)/M_(LNYV)VLPs determined by ImageJ software (NIH, Bethesda, Md.) was 50.5±15 nm.The HAi-TM(G)-M_(LNYV) VLPs were substantially uniform in size andshape.

All documents, books, manuals, papers, patents, published patentapplications, guides, abstracts, and other references cited herein areincorporated by reference in their entirety. Other embodiments of theinvention will be apparent to those skilled in the art fromconsideration of the specification and practice of the inventiondisclosed herein. It is intended that the specification and examples beconsidered as exemplary only, with the true scope and spirit of theinvention being indicated by the following claims.

1. A virus-like particle comprising a matrix protein derived from afirst plant enveloped virus and a surface polypeptide, wherein thesurface polypeptide comprises (a) a surface exposed portion derived froma target polypeptide, (b) a transmembrane domain, and (c) a cytosolictail derived from a transmembrane protein of a second plant envelopedvirus.
 2. The virus-like particle of claim 1, wherein the first plantenveloped virus and the second plant enveloped virus are the same. 3.The virus-like particle of claim 1, wherein the virus-like particle isproduced in a plant cell, a plant, or a portion of a plant.
 4. Thevirus-like particle of claim 1, wherein the first plant enveloped virusis a plant rhabdovirus.
 5. The virus-like particle of claim 4, whereinthe plant rhabdovirus is selected from the group consisting of LettuceNecrotic Yellows virus (LNYV), Northern Cereal Mosaic virus (NCMV),Sonhus Virus (SonV), and Broccoli necrotic yellows virus (BNYV).
 6. Thevirus-like particle of claim 1, wherein the surface exposed portion ofthe surface polypeptide is antigenic.
 7. The virus-like particle ofclaim 1, wherein the target polypeptide is derived from a pathogen. 8.The virus-like particle of claim 1, wherein the surface exposed portionof the surface polypeptide is therapeutic.
 9. The virus-like particle ofclaim 1, wherein the target polypeptide is a native surface polypeptide.10. The virus-like particle of claim 1, wherein the target polypeptideis an artificial surface polypeptide.
 11. The virus-like particle ofclaim 1, wherein the transmembrane domain is native or foreign to thesurface exposed portion.
 12. The virus-like particle of claim 1, whereinthe transmembrane domain is derived from the transmembrane protein. 13.The virus-like particle of claim 1, wherein the transmembrane domain isderived from a cellular or viral transmembrane protein.
 14. A method ofproducing virus-like particles in a plant cell, a plant or a portion ofa plant, comprising (a) introducing one or more nucleic acid moleculesinto the plant cell, the plant or the portion of a plant, wherein theone or more nucleic acid molecules comprise a first nucleotide sequenceencoding a matrix protein derived from a first plant enveloped virus anda second nucleotide sequence encoding a surface polypeptide, wherein thesurface polypeptide comprises (i) a surface exposed portion derived froma target polypeptide, (ii) a transmembrane domain, and (iii) a cytosolictail derived from a transmembrane protein of a second plant envelopedvirus; and (b) maintaining the plant cell, the plant or the portion of aplant under conditions permitting co-expression of the matrix proteinand the surface polypeptide, whereby the virus-like particles areproduced.
 15. The method of claim 14, wherein the first plant envelopedvirus and the second plant enveloped virus are the same.
 16. The methodof claim 14, wherein the virus-like particles are substantially uniformin size.
 17. The method of claim 14, further comprising (c) purifyingthe virus-like particles.
 18. The method of claim 14, wherein the one ormore nucleic acid molecules are introduced into the plant cell, theplant or the portion of a plant by infiltration, particle bombardment,or inoculation.
 19. The method of claim 14, wherein the one or morenucleic acid molecules are introduced into the plant cell, the plant orthe portion of a plant transiently or stably.
 20. An immunogeniccomposition comprising an effective amount of the virus-like particlesof claim 1, wherein the virus-like particles are substantially uniformin size.
 21. An immunogenic composition comprising an effective amountof the virus-like particles produced by the method of claim 14, whereinthe virus-like particles are substantially uniform in size.
 22. Theimmunogenic composition of claim 20, further comprising an adjuvant oran excipient.
 23. A method of inducing a protective immune response to apathogen in a subject, comprising administering to the subject aneffective amount of the virus like particles produced by the method ofclaim 14, wherein the target polypeptide is derived from the pathogen,and wherein the virus-like particles are substantially uniform in size.24. A recombinant plant cell comprising one or more nucleic acidmolecules, wherein the one or more nucleic acid molecules comprise afirst nucleotide sequence encoding a matrix protein derived from a firstenveloped virus and a second nucleotide sequence encoding a surfacepolypeptide, and wherein the surface polypeptide comprises (a) a surfaceexposed portion derived from a target polypeptide, (b) a transmembranedomain, and (c) a cytosolic tail derived from a glycoprotein of a secondenveloped virus.
 25. The recombinant plant cell of claim 24, wherein thefirst plant enveloped virus and the second plant enveloped virus are thesame.
 26. A plant or a portion thereof comprising the plant cell ofclaim 24.